Project summary Hepatitis C virus (HCV) is an important and underreported infectious disease, causing chronic infection in ~71 million people worldwide. The CDC estimates there are ~30,000 new cases of HCV every year in the US and about 20,000 deaths annually, making HCV more deadly than 60 other infectious diseases combined, including HIV. However, the underlying mechanisms that lead to chronic HCV infection followed by end-stage liver disease are poorly understood. Although chronic hepatitis C can now be effectively treated with direct-acting antivirals (DAAs), a vaccine to prevent transmission remains a high priority due to extremely high treatment costs ($80,000+ for a 12-week course). Furthermore, once infected, individuals remain at high risk for liver disease even post-treatment. The proposed work will build on our substantial research findings to continue systematically analyzing the mechanisms that create barriers to interspecies HCV transmission. Aim 1: Define mechanisms of known host factors from diverse species that support HCV replication. HCV relies on a variety of host factors to establish replication in hepatocytes. Our preliminary data demonstrate that for all great apes tested, orthologs of peptidylprolyl isomerase A, also known as cyclophilin A (CypA), support HCV RNA replication. However, CypA of distantly related species, such as New and Old World monkeys and mice, is far less efficient. We aim to define mechanistically the underlying incompatibility of this and other host factors with the virally encoded components of the HCV replication machinery. Aim 2: Characterize HCV infection and immune response networks following infection across primate and rodent species. We will study whether HCV can infect and replicate in a novel set of stem cell-derived hepatocyte-like cells from a range of evolutionarily diverse species, including great apes, selected New and Old World monkeys and rodents. In a second step, we will use high-throughput single-cell RNA sequencing to derive species-specific transcriptomic response networks associated with HCV replication. Aim 3: Characterize the impact of CD302 and Cr1L on restricting HCV infection in vivo. Human entry factor transgenic mice with blunted innate immunity only support low-level viral replication, suggesting murine restriction factors may suppress viral replication in vivo. Our preliminary data indicate mouse CD302 and complement component (3b/4b) receptor 1-like (Cr1L) limit HCV replication in vitro. Here, we will assess if loss- of-function of mCD302 and mCr1L augments HCV infection in our HCV entry factor knock-in mice. The proposed work will provide new insights into the species tropism of HCV. The Ploss lab has made many seminal contributions to the field and will be aided in this important work by our long-standing collaborators Drs. Schwartz (Weill Cornell), Shalek (MIT) and Pietschmann (Twincore, Germany). Our work will advance the field of HCV research by making progress in the development of small animal models suitable for studying HCV infection and immune responses, a necessary precursor to improving treatment and developing vaccines.